Amyloid inhibitory peptides

ABSTRACT

The present invention relates to peptides, in particular amyloid inhibitory peptides, and to pharmaceutical compositions comprising such peptides. Furthermore, the present invention relates to such peptides, in particular such amyloid inhibitory peptides, for use in methods of treating or diagnosing neurodegenerative diseases such as Alzheimer&#39;s disease, or for use in a method of treating or diagnosing type 2 diabetes. Furthermore, the present invention also relates to a kit for the in-vitro or in-vivo detection and, optionally, quantification of amyloidogenic polypeptides, amyloid fibrils or amyloid aggregates, and/or for the diagnosis of Alzheimer&#39;s disease or type 2 diabetes in a patient.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a National Stage Application of InternationalApplication Number PCT/EP2019/064793, filed Jun. 6, 2019; which claimspriority to European Application No. 18176609.8, filed Jun. 7, 2018.

The Sequence Listing for this application is labeled“SeqList-14Jan21-ST25.TXT”, which was created on Jan. 14, 2021 and is 10KB. The entire content is incorporated herein by reference in itsentirety.

The present invention relates to peptides, in particular amyloidinhibitory peptides, and to pharmaceutical compositions comprising suchpeptides. Furthermore, the present invention relates to such peptides,in particular such amyloid inhibitory peptides, for use in methods oftreating or diagnosing neurodegenerative diseases such as Alzheimer'sdisease, or for use in a method of treating or diagnosing type 2diabetes. Furthermore, the present invention also relates to a kit forthe in-vitro or in-vivo detection and, optionally, quantification ofamyloidogenic polypeptides, amyloid fibrils or amyloid aggregates,and/or for the diagnosis of Alzheimer's disease or type 2 diabetes in apatient.

Amyloid self-assembly is linked to devastating cell-degenerativediseases including Alzheimer's disease (AD) and type 2 diabetes (T2D).Molecules blocking amyloidogenesis of the key amyloid polypeptides of ADand T2D amyloid-β peptide (Aβ40(42)) (AD) and islet amyloid polypeptide(IAPP) (T2D) could thus become drug candidates. However, the rationaldesign of amyloid inhibitors is a difficult task. Major reasons are thehigh conformational flexibility of most amyloidogenic polypeptides, highaffinity interactions of amyloid self-assembly, and the large size ofinvolved interfaces. Additional challenges include a lowblood-brain-barrier (BBB) permeability, high production costs, andpotential immunogenicity of antibodies, low proteolytic stability andusually no BBB crossing of linear peptides, while small molecules oftenlack high affinity and specificity and cannot block interactionsinvolving large interfaces. Importantly, none of the reported inhibitorsof amyloid self-assembly of Aβ40(42) or IAPP has yet advanced to theclinic.

Accordingly, there is a need for new inhibitors of amyloid self-assemblyof Aβ40(42) or IAPP. There is furthermore a need to provide amyloidinhibitors that are specific for amyloid self-assembly of Aβ40(42).

In a first aspect, the present invention relates to a peptide,preferably an amyloid inhibitory peptide, having an amino acid sequenceaccording to formula 0

whereinZ1 and Z2 are selected from the following pairs

-   -   a) cysteine and cysteine,    -   b) aspartic acid and lysine, or lysine and aspartic acid,    -   c) aspartic acid and ornithine, or ornithine and aspartic acid,    -   d) aspartic acid and 2,4-diaminobutyric acid, or        2,4-diaminobutyric acid and aspartic acid,    -   e) aspartic acid and 2,3-diaminopropionic acid, or        2,3-diaminopropionic acid and aspartic acid, f) glutamic acid        and lysine, or lysine and glutamic acid,    -   g) glutamic acid and ornithine, or ornithine and glutamic acid,    -   h) glutamic acid and 2,4-diaminobutyric acid, or        2,4-diaminobutyric acid and glutamic acid,    -   i) glutamic acid and 2,3-diaminopropionic acid, or        2,3-diaminopropionic acid and glutamic acid;        with        denoting a covalent bond between Z1 and Z2, thus providing for a        cyclization of the peptide;        X1, X2, X3, X4, X5, X6, and X7 are, independently at each        occurrence, selected from glycine, asparagine, valine,        histidine, leucine, serine, alanine, and threonine;        F is, independently at each occurrence, phenylalanine;        L is leucine;        U is, independently at each occurrence, selected from arginine,        homoarginine, citrulline, ornithine, lysine, and norleucine;        G is glycine;        I is isoleucine;        wherein Z1, Z2, X1-X7, F, L, U, G and I are L-amino acid        residues or D-amino acid residues, or some of Z1, Z2, X1-X7, F,        L, U, G and I are L-amino acid residues and others are D-amino        acid residues;        and pharmaceutically acceptable salts, esters, solvates,        polymorphs and modified forms thereof;        wherein preferably said peptide has an amino acid sequence        according to formula 0a

whereinZ1, Z2, X1-X7, F, L, U, G, I are as defined above, and

In one embodiment, the peptide according to the present invention,preferably the amyloid inhibitory peptide according to the presentinvention, has an amino acid sequence according to formula 1

or an amino acid sequence according to formula 1*

whereinC is cysteine;X1, X2, X3, X4, X5, X6, and X7 are, independently at each occurrence,selected from glycine, asparagine, valine, histidine, leucine, serine,alanine, and threonine;F is, independently at each occurrence, phenylalanine;L is leucine;R is arginine;G is glycine;I is isoleucine;

is a disulfide bond;

is N-methyl;C, X1-X7, F, L, R, G and I are L-amino acid residues or D-amino acidresidues, or some of C, X1-X7, F, L, R, G and I are L-amino acidresidues and others are D-amino acid residues;and pharmaceutically acceptable salts, esters, solvates, polymorphs andother modified forms thereof.

In an embodiment of the peptide, either

-   -   a) X1 and X4 are asparagine, X2 is valine, X3 is histidine, X4        is glycine, X5 is glycine, X6 and X7 are glycine;    -   b) X1-X7 are glycine, alanine or serine;    -   c) X1-X7 are glycine;    -   d) X1-X3 are glycine, X4 is asparagine, X5 is alanine, and X6-X7        are glycine; or    -   e) X1-X3 are glycine, X4 is asparagine, X5 is alanine, X6 is        leucine, X7 is serine.

In one embodiment, Z1, Z2, C, X1-X7, F, L, U, R, G, and I are L-aminoacid residues.

In one embodiment, R is, at each occurrence, D-arginine, and/or

F is, at each occurrence, D-phenylalanine, and/or L is D-leucine, and/or

Z1, Z2 and C are D-amino acid residues, and/or

I is D-isoleucine or N-methyl-D-isoleucine.

In one embodiment, the peptide according to the present invention has asequence according to a formula selected from the following formulae2a-2e, 2a*-2e*:

wherein upper case letters represent L-amino acid residues or D-aminoacid residues, preferably L-amino acid residues, and lower case lettersrepresent D-amino acid residues.

In a particularly preferred embodiment, the peptide according to thepresent invention has a sequence according to a formula selected from 2eand 2e*

wherein upper case letters represent L-amino acid residues or D-aminoacid residues, preferably L-amino acid residues, and lower case lettersrepresent D-amino acid residues.

In one embodiment, said peptide consists of a sequence according to anyof formulae 0, 0a, 1, 1*, 2a-2e, 2a*-2e*, as defined above,respectively.

In one embodiment, said peptide is an amyloid inhibitory peptide thatpreferably binds to Aβ40(42) and/or to islet amyloid polypeptide (IAPP),or said peptide is a peptide that binds to Aβ40(42) and/or to isletamyloid polypeptide (IAPP), but is not necessarily an amyloid inhibitorypeptide.

It should be noted that in preferred embodiments, where reference ismade to a “peptide” in general, such peptide may also be referred to asan “amyloid inhibitory peptide”. An “amyloid inhibitory peptide” is apeptide that functions as or can be used as an “amyloid inhibitor”.

Without wishing to be bound by any theory, the present inventors believethat the amyloid inhibitory effect of an amyloid inhibitory peptide ismediated by its binding to key amyloid polypeptides, in particular toAβ40(42) and/or to islet amyloid polypeptide (IAPP).

Hence, the term “amyloid inhibitory” as used herein in the context of apeptide, refers to the capability of such peptide to block or inhibitamyloid self-assembly or amyloidogenesis, preferably of the key amyloidpolypeptides, in particular of Aβ40(42) and/or of islet amyloidpolypeptide (IAPP).

Amyloid inhibitory peptides in accordance with the present invention areuseful as amyloid inhibitors, and may thus be used for therapeuticand/or diagnostic purposes, i.e. they may be used for treatment and/ordiagnosis of diseases involving amyloid self-assembly oramyloidogenesis, in particular of Alzheimer's disease and/or of type 2diabetes.

In other embodiments, a peptide in accordance with the present inventionmay have the capability to bind to key amyloid polypeptides, but may notnecessarily be capable of functioning as an amyloid inhibitor. Suchpeptides may herein also sometimes be referred to as “amyloid bindingpeptides”. They bind to key amyloid peptides, but are not capable ofblocking or inhibiting amyloid self-assembly or amyloidogenesis. Theymay nevertheless be useful for detection purposes, in cases wheredetection of amyloid peptides may be desirable.

In a further aspect, the present invention also relates to a compositioncomprising a peptide, preferably an amyloid inhibitory peptide,according to the present invention, and a suitable solvent, such aswater, and a buffer.

In a further aspect, the present invention also relates to apharmaceutical composition comprising a peptide, preferably an amyloidinhibitory peptide, according to the present invention and apharmaceutically acceptable excipient.

In such pharmaceutical composition, the peptide may occur as such, or itmay be linked to other entities/molecules that endow the peptide with aspecific functionality. For example, there may be a tag attached toincrease blood-brain-barrier permeability, or it may be attached to aspecific reporter molecule, such as a dye or a quantum dot, allowing thedetection in diagnostic methods (preferably whilst retaining thetherapeutic functionality of the peptide—“theranostic applications”).The use of quantum dots may be particularly useful in various imagingtechnologies, such as MRI, PET, PET-MRI, or specificallyquantum-dot-based brain imaging methodologies. In certain embodiments,the peptide may be attached to a nanoparticle, to a suitable carriermolecule, to a targeting entity or other functional molecule.

Because the peptides according to the present invention are capable ofpassing the blood-brain-barrier, the present invention also relates tothe use of a peptide according to the present invention, as definedabove, as a carrier for molecules, substances or compounds to pass theblood-brain-barrier. According to this aspect, in certain embodiments,the molecule to pass the blood-brain-barrier is linked, preferablycovalently linked, to a peptide according to the present invention asdefined above.

In a further aspect, the present invention also relates to a peptide, inparticular an amyloid inhibitory peptide, according to the presentinvention, or the pharmaceutical composition according to the presentinvention as defined above, for use in a method of treating Alzheimer'sdisease or for use in a method of treating type 2 diabetes.

In one embodiment of such peptide or composition for use, said methodcomprises administering an effective amount of said peptide or of saidcomposition to a patient in need thereof.

In a further aspect, the present invention also relates to a peptide, inparticular an amyloid inhibitory peptide, according to the presentinvention, or the pharmaceutical composition according to the presentinvention as defined above, for use in a method of diagnosingAlzheimer's disease or for use in a method of diagnosing type 2diabetes.

In one embodiment of such peptide or composition for use, said methodcomprises administering an effective amount of said peptide or of saidcomposition to a subject to be tested for Alzheimer's disease or type 2diabetes.

In one embodiment of such peptide or composition for use, said peptide,in particular said amyloid inhibitory peptide, is linked to oradministered together with a suitable reporter molecule that allowsdetection of Aβ40(42), islet amyloid polypeptide (IAPP) and/or amyloidaggregates thereof, by a suitable detection methodology, such aspositron emission tomography (PET), nuclear magnetic resonance (NMR),magnetic resonance imaging (MRI), and PET-MRI and said subject, afteradministration of said peptide, is subjected to PET, NMR, MRI, PET-MRI.

In a yet a further aspect, the present invention relates to a kit forthe in-vitro or in-vivo detection of amyloid fibrils or aggregatesand/or for the quantification thereof, or for the diagnosis ofAlzheimer's disease or type 2 diabetes in a patient, said kit comprisingthe peptide, in particular the amyloid inhibitory peptide according tothe present invention as defined above, in a freeze-dried form in asuitable container, a buffered solvent in a separate container forreconstitution of said peptide in solution, and, optionally, means todispense said peptide once reconstituted in solution, such as a syringeor pipette. Alternatively, the kit may contain the peptide, inparticular the amyloid inhibitory peptide according to the presentinvention as defined above, in an already reconstituted, ready-to-useform.

In a yet a further aspect, the present invention also relates to the useof the peptide according to the present invention, in an in-vitro assay,such as an enzyme linked immunosorbent assay (ELISA) or a radioimmunoassay (RIA), for the detection of monomeric islet amyloid polypeptide(IAPP), monomeric Aβ40(42), amyloid fibrils, or amyloid aggregates. Suchuse may, in certain embodiments, involve the analysis of blood,cerebrospinal fluid, or brain biopsies, and may also further involve theuse of suitable reporter molecules to which the peptide may be attachedor with which the peptide may be used together.

In a further aspect, the present invention relates to the use of apeptide according to the present invention, as defined above, for themanufacture of a medicament for the treatment or diagnosis ofAlzheimer's disease or of type 2 diabetes.

In yet a further aspect, the present invention also relates to a methodof treatment or diagnosis of Alzheimer's disease or type 2 diabetes,wherein said method comprises administering an effective amount of saidpeptide or of said composition according to the present invention asdefined further above, to a patient in need thereof or to a subject tobe tested.

The present inventors have provided cyclic peptides which function asnanomolar inhibitors of amyloid self-assembly of both Aβ40(42) or IAPP,or of Aβ40(42) alone, and which therefore have manifold applications.

Moreover, several of these peptides bind, with high affinity Aβ40(42)and/or IAPP monomers and/or amyloid aggregates.

In the present application, use is made of the one-letter-code for aminoacid residues and the three-letter-code for amino acid residues. Hence,amino acid residues are designated herein by reference to theirrespective one-letter-code or three-letter-code. Accordingly, alanine isA or Ala; arginine is R or Arg; asparagine is N or Asn; aspartic acid isD or Asp; cysteine is C or Cys; glutamine is Q or Gln; glutamate is E orGlu; glycine is G or Gly; histidine is H or His; isoleucine is I or Ile;leucine is L or Leu; lysine is K or Lys; methionine is M or Met;phenylalanine is F or Phe; proline is P or Pro; serine is S or Ser;threonine is T or Thr; tryptophan is W or Trp; tyrosine is Y or Tyr;valine is V or Val.

Sometimes, in this application, reference to amino acid sequences ismade by reciting the individual residues. Where such amino acid sequenceis indicated by using upper case letters only, this means that theseamino acids are unspecified in terms of their chirality, i.e. theresidues may be L-amino acids or D-amino acids or a mixture of the twopossibilities, i.e. some of the residues in the sequence may be L-aminoacids and others may be D-amino acids. In one embodiment, the upper caseamino acids may be all L-amino acids.

In those instances, where such amino acid sequence is indicated by usingupper case letters and lower case letters together in one sequence, thismeans that the upper case amino acids may be L-amino acids or D-aminoacids, preferably L-amino acids, and the lower case amino acids are, inany case, D-amino acids.

Moreover, sometimes, in this application, reference to amino acidsequences is made by reciting the individual residues as free aminoacids, such as “glycine”, “glutamic acid”, etc., notwithstanding thefact that these residues appear in the respective amino acid sequence intheir respective covalently linked form, i.e. with the individualresidues linked by appropriate peptide bonds, i.e. amide bonds, betweenthem.

The term “N-methyl” or “NMe” or

as used herein, refers to a methyl group that is attached to thenitrogen in the amide bond between two amino acid residues.

For example where a sequence is indicated as

this means that a methyl group is attached to the amide nitrogen formingthe amide bond between F and G, and a further methyl group is attachedto the amide nitrogen forming the amide bond between X and I. This mayalso be referred to as “N-methylated glycine” and “N-methylatedisoleucine” respectively, or “methylated glycine” and a “methylatedisoleucine”, respectively, because the respective amide nitrogen belongsto glycine and isoleucine respectively in these cases.

In some preferred embodiments of the peptide according to the presentinvention, there is a methyl group attached to the amide bond betweenF11 and G12 (i.e. a “methylated glycine 12”), and a further methyl groupattached to the amide bond between X13 and I14 (i.e. a “methylatedisoleucine 14”), if one uses a numbering which is based on a 17-peptideaccording to any of formulae 0, 0a, 1, 1*, 2, 2a-2e, 2a*-2e*. Byreference to the IAPP-numbering, these positions correspond to the amidebond between F23 and G24 and the amide bond between A25 and 126.

The symbol

refers to a covalent bond between two residues thus linked. For example,if the two residues thus linked are two cysteines, it means a disulfidebond.

Alternatively, it may mean a lactam bridge involving the sidechains ofthe respective two amino acids. For example, the sidechains of asparticacid and lysine, or of glutamic acid and lysine may form such a lactambridge. Pairs that may be involved in such lactam bridge formation areAsp-Lys, Asp-Orn, Asp-Dab (Dab meaning 2,4 diaminobutyric acid), Asp-Dap(Dap meaning 2,3-diaminopropionic acid), Glu-Lys, Glu-Orn, Glu-Dab,Glu-Dap, or pairs with an inverse arrangement of the aforementionedresidues. In one embodiment, it also possible to obtain a cyclisationvia the generation of 1,2,3-triazole rings, generated by click reactionsbetween specific amino acids, instead of cysteines.

In one embodiment, the N-terminus and/or the C-terminus of the peptidesaccording to the present invention are protected. Suitable protectinggroups are manifold and are known to a person skilled in the art. Forexample, the N-terminus may be acetylated or formylated, or there may bean even longer chain attached such as palmitoyl. The C-terminus could beprotected via formation of an amide or carbonic acid ester. In oneembodiment, the C-termini of the peptide(s) according to the presentinvention, in particular of the amyloid inhibitory peptides according tothe present invention, more particularly of the peptides according toformulae 0, 0a, 1, 1*, 2a-2e, 2a*-2e* according to the presentinvention, are protected by an amide. In other embodiments, theC-terminus is in its free carboxy-form; in yet other embodiments, it isin esterified form. In particularly preferred embodiments, the C-terminiof the peptide(s) according to the present invention, in particular ofthe amyloid inhibitory peptides according to the present invention, moreparticularly of the peptides according to formulae 0, 0a, 1, 1*, 2a-2e,2a*-2e* according to the present invention, are protected by an amide,and the respective N-termini of the peptides are unprotected, i.e. intheir NH₂-form (or NH₃ ⁺-form).

The term “treatment” or “treating”, as used herein, encompasses bothprophylactic treatment and therapeutic treatment. In a preferredembodiment, it specifically refers to therapeutic treatment.

The present inventors have managed to design peptidic inhibitors ofamyloid self-assembly of both Aβ40(42) and IAPP, or of Aβ40(42) alone.Without wishing to be bound by any theory, the present inventors believethat the peptidic inhibitors according to the present invention mimicIAPP interaction surfaces while maintaining only minimal IAPP-derivedself/cross-recognition elements. Most interestingly, a switching ofchiralities of certain residues led to an inhibitor that wasspecifically selective for Aβ40(42) and which, in addition, exhibitedboth strongly improved proteolytic stability in human plasma andblood-brain-barrier crossing ability in a cell model.

Furthermore, reference is made to the figures, wherein

FIG. 1 shows an embodiment of the inhibitor design strategy employed bythe present inventors. a) shows the sequences of Aβ40(42) and IAPP. b)shows a partial sequence from IAPP, R3-GI (1) (a peptide based onresidues 8-28 of IAPP) and designed peptides with lower-case-lettersdenoting D-amino acid residues. There are amide bond N-methylations atG12 and I14.

FIG. 2 shows the effects of effects of peptides 1-2a on amyloidself-assembly of Aβ40 and IAPP and CD studies on their conformations. a)Fibrillogenesis of Aβ40 (16.5 μM) or its mixtures (Aβ40/peptide, 1/1 or1/2 (2)) by ThT binding (means (±SD), 3 assays). b) Effects on cellviability: Solutions from 1a (7 day-aged) added to PC12 cells; celldamage determined by MIT reduction (means (±S.D.), 3 assays (n=3 each)).c) Fibrillogenesis of IAPP (6 μM) and its mixtures (IAPP/peptide, 1/2(1a, 2a) or 1/10 (1, 2) by ThT binding (means (±S.D.), 3 assays). d)Cell-damaging effects of IAPP or its mixtures (from 1c; 7 day-aged) onRIN5fm cells via MIT reduction (means (±S.D.), 3 assays (n=3 each)). e)TEM images of Aβ40, IAPP and their mixtures with is and 2a (7 days aged)(bars wo nm). f) CD spectra (5 μM, pH 7.4).

FIG. 3 shows effects of peptides 2b and 2e versus 2a on fibrillogenesisand cytotoxicity of Aβ40 and IAPP and CD studies on inhibitorconformations. a) Fibrillogenesis of Aβ40(16.5 μM) or its mixtures with2a (1/1) and 2b or 2e (1/5) by ThT binding (means (±S.D.), 3 assays). b)PC12 cell viability following treatment with 7 day-aged solutions (from2a) by MIT reduction (means (±S.D.), 3 assays (n=3 each)). c)Fibrillogenesis of IAPP (6 μM) or its mixtures with 2a (1/2) and 2b or2e (1/10) determined by ThT binding (means (±S.D.), 3 assays). d) RIN5fmcell viability following treatment with 7 day-aged solutions (from 2c)by MIT reduction (means (±SD), 3 assays (n=3 each)). e) TEM images ofAβ40 or IAPP and their mixtures with 2b and 2e (7 day-aged) (bars 100nm). f) CD spectra (5 μM, pH 7.4).

FIG. 4 shows proteolytic stabilities of inhibitors in human blood plasmain vitro (a), effects on Aβ42 amyloid self-assembly (b,c) and onAβ42-induced impairment of synaptic LTP ex vivo (d,e), and permeabilityacross a human BBB model in vitro (f,g). a) Peptides were incubated inplasma and quantified by HPLC and MALDI-TOF-MS; remaining intact peptide(% of total) is plotted over incubation time. b) Fibrillogenesis of Aβ42(16.5 μM) or its mixtures with peptides (1a & 2a, 1/1; 2b & 2e, 1/5) byThT binding (means (±SD), 3 assays). c) PC12 cell viability aftertreatment with 7 day-aged solutions from 3b by MIT reduction (means(±SD), 3 assays (n=3)). d,e) Amelioration of Aβ42-induced (50 nM) LTPimpairment in murine acute hippocampal slices by 2b (d) or 2e (e) (500nM). Time course of synaptic transmission (means (±SEM), n=7(Aβ42/inhibitor mixtures), n=4 (Aβ42) and n=9-11 (control). Inset: LTPin presence of Aβ42, Aβ42/inhibitor mixture, or inhibitor alone(control) as above; bars show average from the last 5 min of recordings(means (±SEM), n=7-14); * indicates significant effects for the mixturesor controls versus Aβ42 (p<0.05, n=7-11 (unpaired t-test). f) Relativepermeability of 2e (Fluos-2e; 10 μM) across the BBB cell model; plotshows Fluos-2e amount in acceptor chamber at various time points (means(±SEM), n=3-4). g) RP-HPLC chromatogram of solution in acceptor chamberat 2 h (from 3f) (left) and MALDI-TOF spectrum (right) of peptide elutedat ˜18 min (*, Fluos-2e, calculated [M+H]⁺=2053.20, found 2053.38);additional peaks are from assay medium.

Moreover, reference is made to the following examples which are given toillustrate and not to limit the present invention.

EXAMPLES Example 1—Materials and Methods

Peptides and Peptide Synthesis

Aβ40 (TFA salt) was synthesized by Fmoc-based solid phase synthesis(SPPS) on Tentagel R PHB resin (Rapp Polymere) using previouslypublished protocols, purified by RP-HPLC and treated as described. Aβ40stocks were freshly prepared in 1,1,3,3,3,3-hexafluoro-2-isopropanol(HFIP) (4° C.); their concentrations were determined by thebicinchoninic acid (BCA) assay (Pierce). IAPP was synthesized by SPPSand the Fmoc-strategy on RINK resin, subjected to air oxidation andpurified by RP-HPLC. Freshly made IAPP stocks in HFIP (200-500 μM) werefiltered over 0.2 μm filters (Millipore) (4° C.); concentrations weredetermined by UV spectroscopy. N^(a)-terminal fluorescein-labeled IAPP(Fluos-IAPP) and N-terminal 7-diethylaminocoumarin-3-carbonyl-labeledAβ40 (Dac-Aβ40) for fluorescence spectroscopic titrations weresynthesized by Fmoc-based SPPS, purified and handled. Synthetic Aβ42(TFA salt) was from PSL (Heidelberg) and its HFIP stocks were made.Synthetic N-terminal FITC-β-Ala-labeled Aβ42 (FITC-Aβ42) (FITC,fluorescein isothiocyanate) for fluorescence titrations was from Bachem;its HFIP stock solutions were always freshly made (4° C.) and theirconcentrations determined by UV spectroscopy. ISMs, MCIPs, controlpeptides (analogs of 2a and 2b), THRPPMWSPVWP-amide (Trfb) and theirN-terminal fluorescein-labeled analogs (C-terminal amides) weresynthesized by Fmoc-based SPPS on Rink-resin and cleaved from the resinusing previously published protocols. Disulphide bridge formation ofMCIPs and control peptides was performed by dissolving crude peptide(after cleavage and lyophilization) at 1 mg/ml in aqueous 0.1 M NH₄HCO₃solution containing 40% DMSO; the progress of the oxidation reaction wasfollowed by RP-HPLC. Peptides were purified by RP-HPLC using previouslydescribed protocols and characterized by MALDI-TOF mass spectrometry(MS) Stock solutions of all peptides were freshly made in HFIP (4° C.)and concentrations were determined by peptide weight and by UVspectroscopy in the case of fluorescently labeled peptides.

Thioflavin T (ThT) Binding Assays

To study the effects of peptides on Aβ40(42) and IAPP fibrillogenesispreviously established ThT binding assay systems were used. Briefly,Aβ40 (16.5 μM) or IAPP (6 μM) and their mixtures with peptides wereincubated for up to 7 days in ThT assay buffer at room temperature. TheThT assay buffer consisted of aqueous 50 mM sodium phosphate buffer, pH7.4, containing 100 mM NaCl and 1% HFIP (Aβ40(42) related studies) or0.5% HFIP (IAPP related studies). Each experimental set containedincubations of Aβ40(42) or IAPP alone as controls. At indicated timepoints, aliquots were gently mixed with a ThT solution (20 μM ThT in0.05 M glycine/NaOH, pH 8.5); ThT binding was determined immediately bymeasuring fluorescence emission at 486 nm upon excitation at 450 nmusing a 2030 Multilabel Reader VictorX3 (PerkinElmer Life Sciences).Effects of peptides added at specific pre- and post-nucleation timepoints of Aβ40 amyloidogenesis were studied by adding aliquots of Aβ40solutions (16.5 μM; incubation conditions as above) aged for theindicated time points to the peptide (in dry form) as previouslydescribed and ThT binding was determined as above. To determine theeffects of peptides on already nucleated IAPP fibrillogenesis, aliquotsof IAPP (16.5 μM; incubation conditions as above), which was aged forthe indicated time points and contained significant amounts of IAPPfibrils (as confirmed by ThT binding and TEM (data not shown)) was addedto the peptide (in dry form) as described and ThT binding was determinedas above.

Transmission Electron Microscopy (TEM)

10 μl aliquots of solutions of the ThT binding and MTT assays wereapplied on carbon-coated grids at indicated time points, washed withdistilled water and stained with aqueous 2% (w/v) uranyl acetate asdescribed. Grids were examined using a JEOL JEM 100CX (at 100 kV) or aJEOL 1400 Nus electron microscope (at 120 kV).

Assessment of Cell Damage by MTT Reduction Assay

Effects of peptides on the formation of cell-damaging IAPP or Aβ40(42)assemblies were studied using the solutions applied for the ThT bindingassays as previously described. Briefly, for effects on the formation ofcell-damaging Aβ40(42) aggregates, the inventors used cultured PC-12cells while for effects on IAPP-mediated cell damage cultured RIN5fmcells were used. Both cell lines were cultured and plated in 96-wellplates as described. Solutions consisting of Aβ40(42) or IAPP alone(16.5 and 6 μM, respectively) versus their mixtures with the potentialinhibitors were made as described under “ThT binding assays” andincubated for 7 or 8 days at room temperature. At the incubation timepoints of 24 h or 72 h and 7 (or 8) days (identical results wereobtained from solutions aged for 7 or 8 days), aliquots were dilutedwith cell culture medium and added to the cells at the indicated finalconcentrations. Following incubation with the cells for ˜20 h (37° C.,humidified atmosphere with 5% CO₂), the MTT reduction assay was used toassess cell damage/metabolic activity as previously described. Todetermine IC₅₀ values, Aβ40 (500 nM) or IAPP (100 nM) were titrated withdifferent amounts of peptides under the conditions of the ThT bindingassays and cell-damaging effects were determined using 24 h-agedsolutions (IAPP-related effects) or 72 h-aged solutions (Aβ40-relatedeffects) by the MIT assay as described. Of note, studies with selectedMCIPs (incubated under the same conditions as in their mixtures withAβ40(42) or IAPP) showed that they were, as expected, non-amyloidogenicand not cytotoxic (data not shown). These results were in line with thefact that the sequences of the MCIPs were derived from thenon-amyloidogenic and non-cytotoxic ISM R3-GI and with results ofprevious studies showing the lack of amyloidogenicity and cell-damagingeffects of related N-methylated IAPP analogs or segments.

To determine the effects of peptides on preformed cell damagingassemblies of Aβ40 or IAPP, aliquots of Aβ40 or IAPP solutions (16.5 μM;incubation conditions as above), aged for the indicated time points andcontaining significant amounts of cell damaging assemblies (as shown bythe MIT reduction assay in this and previous studies (data not shown))were added to the peptide (in dry form) as described and followingincubation for the indicated time points solutions were added to PC12 orRIN5fm cells. Following incubation with the cells for ˜20 h, the MITreduction assay was performed as described. Of note, our assay systemallows following in parallel formation of both fibrils (by the ThTbinding assay) and cell damaging assemblies of Aβ40(42) and IAPPstarting from non-fibrillar and non-toxic states as previouslydescribed.

Far-UV CD Spectroscopy

Far-UV CD measurements were performed with a Jasco 715spectropolarimeter as described. Spectra were measured immediatelyfollowing solution preparation between 195 and 250 nm, at 0.1 nmintervals, a response time of 1 sec, each spectrum being the average of3 spectra and at room temperature. CD measurements were performed usingfreshly made 5 μM solutions of ISMs in aqueous 10 mM sodium phosphatebuffer, pH 7.4, containing 1% HFIP (CD assay buffer); peptides werediluted from freshly made stock solutions in HFIP into the aqueous assaybuffer. Of note, the magnitudes of the CD spectra of allpeptides/inhibitors depend on their concentrations due to the inherentlystrong self-association potential of peptides derived from the humanIAPP sequence (data not shown). The spectrum of the buffer wassubtracted from the CD spectra of the peptide solutions prior conversionof the raw data to mean residue ellipticities.

Fluorescence Spectroscopic Titration Studies

A JASCO FP-6500 fluorescence spectrophotometer was used for thefluorescence spectroscopic titrations, which were performed by usingpreviously described experimental protocols. Briefly, for titrations ofFluos-IAPP and FITC-labeled Aβ42, excitation was at 492 nm andfluorescence emission spectra were recorded between 500 and 600 nm,while for titrations of Dac-Aβ40, excitation was at 430 nm and emissionspectra were collected between 440 and 550 nm. App. K_(d)s ofinteractions of IAPP, Aβ40 and Aβ42 with the peptides were determined bytitrating freshly made solutions of Fluos-IAPP (5 nM), Dac-Aβ40(10 nM)and FITC-Aβ42 (5 nM) with peptides as described. Of note, this assaysystem has been previously used for the determination of the affinities(app. K_(d)) of interactions of a number of inhibitors of IAPP andAβ40(42) amyloid self-assembly with these highly amyloidogenicpolypeptides and the affinity of the Aβ40(42)-IAPP interactions as well.Briefly, freshly made stock solutions of peptides and fluorescentlylabeled analogs in HFIP were used. Measurements were performed in 10 mMsodium phosphate buffer, pH 7.4 (i % HFIP) at room temperature within2-5 min following solution preparation. Under these experimentalconditions, freshly made solutions of Fluos-IAPP, Dac-Aβ40 and FITC-Aβ42at the herein-applied low nanomolar concentrations contain mostlymonomers. App. K_(d)s were estimated using 1/1 binding models aspreviously described. Of note, due to the inherently high self-assemblypotentials of IAPP-derived peptides more complex binding models may alsoapply. Determined app. KdDs are means (+SD) from three binding curves.

Cross-Linking, NuPAGE and Western Blot Analysis

Cross-linking studies were performed using a previously establishedassay system used for the characterization of hetero-assemblies of Aβ40and IAPP with IAPP and IAPP-derived inhibitors including ISMs asdescribed.^([1, 4]) Briefly, Aβ40 or IAPP were incubated alone (30 μM)or in the presence of peptides (IAPP/peptide and Aβ40/peptide 1/10) inaqueous 10 mM sodium phosphate buffer, pH 7.4, at room temperature for 3h (Aβ40 related studies) or 30 min (IAPP related studies). Thereafter,samples were cross-linked with aqueous glutaraldehyde (25%)(Sigma-Aldrich) and cross-linked hetero-complexes were precipitated withaqueous trichloroacetic acid (TCA) (10%); pellets were dissolved inNuPAGE sample buffer (w/o reducing agent), boiled (5 min) and NuPAGEelectrophoresis in 4-12% Bis-Tris gels with MES running buffer wasperformed according to the manufacturer's (Invitrogen) recommendations.Equal amounts of Aβ40 or IAPP were loaded in all lanes. For peptideblotting onto nitrocellulose, a XCell II Blot Module blotting system(Invitrogen) was used. Aβ40 and Aβ40-containing complexes were revealedby Western blotting and a polyclonal rabbit anti-Aβ40 antibody(Sigma-Aldrich) while IAPP and IAPP-containing complexes by a polyclonalrabbit anti-IAPP antibody (Bachem) in combination with peroxidase(POD)-coupled secondary antibody (Amersham) and the Super SignalDuration ECL staining solution (Pierce).

Peptide Stability in Human Plasma (In Vitro)

Peptides were dissolved in human blood plasma (obtained from voluntaryhealthy donors) at a concentration of 200 μM and incubated at 37° C. forvarious time intervals. Following quenching (1/1) with aqueoustrichloroacetic acid (10%), solutions were incubated on ice for 10 min,subjected to centrifugation to precipitate plasma proteins (20200 g; 4min), and the supernatants were mixed (1/2) with a solution consistingof 80% HPLC buffer B and 20% HPLC buffer A (see below). To quantifyintact peptide at different time points, solutions containing thesupernatants were analyzed by RP-HPLC (detection at 214 nm) by using aNucleosil 100 C18 column (Grace) (length 33 mm length, ID 8 mm, 7 μmparticle size) with a flow rate of 2.0 ml/min and eluting buffers A,0.058% (v/v) TFA in water, and B, 0.05% (v/v) TFA in 90% (v/v) CH₃CN inwater. The elution gradient was 10-90% B in A over 8 min; this step wasfollowed by a 90-10% B in A over 3 min step to establish startingconditions. HPLC fractions were collected, lyophilized, and analyzed byMALDI-TOF-MS using a Bruker Daltonik MALDI-TOF MS instrument.

Peptide Stability Toward Degradation by Human Neprilysin (In Vitro)

Stabilities of 2e and 2b toward degradation by neprilysin was studiedbased on a previously published protocol: Briefly, peptides wereincubated (100 μM) with recombinant human neprilysin (NEP) (500 ng/ml)(Sigma-Aldrich) in 10 mM Tris buffer, pH 6.5 and at 37° C. At indicatedtime points aliquots were quenched (1/1) with aqueous trichloroaceticacid (10%) and solutions were subjected to HPLC analysis and HPLCfractions were collected and analyzed by MALDI-TOF-MS as described under“Peptide stability in human plasma” (above).

Determination of Surface Neprilysin/CD10 Levels by Flow Cytometry

Both the human cerebral microvascular endothelial cell line hCMEC/D3 andhuman umbilical vein endothelial cells (HUVEC) were cultured on collagentype I-coated plates (BD Biosciences) in EndoGRO™-MV Complete Media Kit(Merck) supplemented with 1 ng/mL fibroblast growth factor-basic (bFGF)(Merck) and maintained at 37° C. in a humidified atmosphere of 5% CO₂.Cell culture medium was routinely replaced every 2-3 days.

The cell surface expression of human CD10, also termed CALLA orneprilysin, on hCMECs and HUVECs was monitored by flow cytometry.Briefly, 1×10⁶ cells were washed three times with ice-coldphosphate-buffered saline (PBS) supplemented with 0.5% bovine serumalbumin (BSA) and subsequently stained with phycoerytherin(PE)-conjugated anti-CD10 antibody (eBioscience) or the correspondingisotype control IgG for 1 h at 4° C. After incubation, the cells werewashed and analyzed by a BD FACSVerse™ flow cytometer (BD Biosciences).The quantification of the measurements was performed using FlowJosoftware.

Hippocampal Long-Term Potentiation (LTP) Measurements (Ex Vivo)

LTP measurements were performed as follows: Briefly, sagittalhippocampal slices (thickness: 350 μm) were obtained from adult (2months) C57/BL6 male mice. Protocols were approved by the ethicalcommittee on animal care and use of the government of Bavaria, Germany.Mice were anaesthetized by inhalation of isoflurane before decapitationand brains were rapidly removed. Hippocampal slices were prepared inice-cold Ringer solution, placed in a holding chamber for at least 90min—the first 30 min at 35° C., the following 60 min cooled down to roomtemperature—and then transferred to an immersion superfusing chamber forextracellular recordings. The flow rate of the solution through thechamber was 5-8 ml/min. The composition of the Ringer solution was 124mM NaCl, 3 mM KCl, 26 mM NaHCO₃, 2 mM CaCl₂, 1 mM MgSO₄, 25 mMD-glucose, and 1.24 mM NaH₂PO₄; solution was bubbled with a mixture of95% O₂ and 5% CO₂, and its pH was 7.3±0.1. Extracellular recordings weremade by glass microelectrodes (2-3 MO filled with artificialcerebrospinal fluid (ACSF) and all measurements were performed at roomtemperature. Synthetic Aβ42 was freshly dissolved in ACSF and added tothe bath solution (50 nM).

Field excitatory postsynaptic potentials (fEPSPs) were evoked bystimulating the Schaffer collateral commissural pathway (Sccp) in thedendritic region of hippocampal CA1 as described. For LTP induction,high-frequency stimulation (HFS; 100 Hz/100 pulses) conditioning pulseswere delivered to the same Sccp inputs. For most recordings, bothstimulating electrodes were used to utilize the input specificity of LTPand allowing the measurement of an internal control within the sameslice. Aβ42 alone (50 nM) was applied for 90 min before high-frequencystimulation (HFS), providing time for its oligomerisation. Mixtures ofAβ42 (50 nM) with each of the tested peptides 2a, 2b and 2e (500 nM)were also applied for 90 min before HFS. Peptides alone (500 nM) wereapplied to the slices 1 h before HFS. Responses were measured for 60 minafter HFS. Recordings were processed and data re-analysed as described.fEPSP slopes measurements were taken between 20 and 80% of the peakamplitude and EPSP slopes are presented as % EPSP slope of baseline (20min control period before tetanic stimulation (100%)). Data wereanalysed by paired t-test.

Human BBB Transwell Permeability Assay (In Vitro)

Human cerebral microvascular endothelial (hCMEC/D3) cells (Merck) werecultured in collagen type I-coated dishes in EndoGro-MV complete culturemedia kit supplemented with 1 ng/mL fibroblast growth factor-2 (FGF-2)(all reagents from Merck) (at 37° C. and 5% CO₂). Of note, confluenthCMEC monolayers in Transwell filters represent a suitable model of thehuman blood-brain-barrier (BBB) with reasonable transendothelialelectrical resistance (TEER) values of 30-100 Ω/cm². Briefly, this cellmodel has been extensively characterized and found to maintain a brainendothelial phenotype; despite lower complexity due to lack ofco-cultured astrocytes/pericytes or application of flow-based shearstress, hCMEC monolayers have suitable barrier characteristics with highjunctional integrity, restricted permeability to paracellular tracers,and reasonable transendothelial electrical resistance (TEER) values of30-100 Ω/cm². The Transwell permeability assay was then performed basedon a previously established assay using 24-well plates (Sigma-Aldrich)containing 6.5 mm Transwell inserts (0.4 μm pore polycarbonate membrane(Corning)) as follows:

hCMEC/D3 cells were grown in endothelial cell medium (ECM), containingEndoGro-MV complete media, 10% fetal bovine serum (FBS), 1%penicillin-streptamycine (P/S), and 1 ng/mL FGF-2 on the membranes ofthe Transwell inserts (2×10⁵ cells/insert) (37° C. and 5% CO₂) untilfull confluency was reached (normally after 5-7 days of culture).Confluency and BBB-type tightness was verified by TEER analysis; TEERvalues of 60-80 Ωcm² were obtained. The upper and lower chambers thenwere reconstituted with 200 μl and 800 μl, respectively, of ECMcontaining 2% FBS. The N-terminal fluorescein-labeled 12 amino acid-longpeptide THRPPMWSPVWP-amide (Fluos-Trfb) known to readily cross the BBBvia binding to the transferrin receptor, was used as a positive controlfor BBB permeability in this model. Synthetic N-terminalfluorescein-labeled 2e (Fluos-2e) (10 μM), Fluos-Trfb (10 μM), orLucifer yellow (20 μM) (control for BBB tightness) were added to theupper (donor) chamber of the Transwell device (n=3 for each incubationtime point and reagent) and incubated (37° C., 5% CO₂) with the cellsfor the indicated time points.

For quantification of fluorescently labeled peptides present in thelower (acceptor) chamber, 100 μl of medium was transferred from thelower chamber to a well of a 96-well black polystyrene plate (Greiner)and fluorescence at 519 nm (excitation at 495 nm) was measured with afluorescence microplate reader (Perkin Elmer Enspire). As a referencepoint for the maximum fluorescence intensity, 100 μl of medium from theupper chamber at 0 h was also transferred and quantified. Of note,fluorescence of Fluos-2e or Fluos-Trfb was directly proportional totheir concentrations as determined by calibration curves. Lucifer yellowin the lower chamber was quantified by measuring the fluorescence at 530nm (excitation at 485 nm). Relative permeability (FIG. 3f ) wascalculated by dividing the fluorescence value of the aliquot from thelower chamber at each time point by the fluorescence value of the 0h-lower chamber aliquot.

Apparent permeability (P_(app)) at 2 h was calculated by the followingequation:P _(app) (cm/s)=(dQ/dt)(1/A)(1/C ₀) (cm/s)where (dQ/dt) is the amount of peptides or Lucifer yellow present in thelower chamber at the 2 hour time point (nmol/s), A is the membrane areaof the upper chamber (0.33 cm²) and C₀ is their initial concentration inthe upper chamber (nmol/ml). Reported P_(app) values are means (±SEM)from at least 3 transport assays (each of them performed intriplicates).

Of note, studies on the transport rate of Lucifer yellow(P_(app)=5.3×10⁻⁶ (±2.7) cm/s at 2 h) were performed in parallel to thestudies on the peptide transport and were consistent with a goodtightness or integrity of the hCMEC monolayer, confirming the TEERmeasurements. In addition, a P_(app) of 20.8×10⁻⁶ (+1.9) cm/s was found(at 2 h) for Fluos-Trfb, which further confirmed the validity of theresults of the permeability assay.

Finally, to confirm the above results and the intact nature of theFluos-2e molecule after crossing the hCMEC BBB-type cell layer, aliquotsof upper and lower chambers were analysed by RP-HPLC and MALDI-TOF-MS(FIG. 4g ). RP-HPLC (detection at λ=214 nm) was performed using a YMCbasic column (length 250 mm; ID 4.6 mm; 5 μm particle size), flow rate1.0 ml/min and eluting buffers A, 0.058% (v/v) TFA in water, and B,0.05% (v/v) TFA in 90% (v/v) CH₃CN in water. The elution gradient was10-90% B in A over 30 min.

Example 2

Design of Peptides and Experiments

Macrocyclic Inhibitory peptides (“MCIPs”) were designed using R3-GI (1),a partial sequence peptide of IAPP (residues 8-28 of IAPP), containingan RRR tripeptide instead of the three residues 19-21 of the IAPPsequence (see also FIG. 1), as a template, conformational restriction bycyclization, sequence truncation, specific N-methylations, and multipleamino acid substitutions with Gly or D-residues (FIG. 1). Notably, 1 ofFIG. 1 is able to inhibit A40(42) but not IAPP amyloidogenesis.

As interaction surfaces mimicking surfaces of β-hairpins/β-sheet foldsof IAPP are likely required for inhibitory function, the presentinventors first asked whether cyclization of partially disordered 1would affect its function and synthesized its cyclic analog is (FIG. 1).Amyloid formation of Aβ40 and IAPP alone or with is were followed by ThTbinding assay and results confirmed by TEM (FIG. 2). In addition, agedsolutions were added to cells and cell-damage studied by the 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reductionassay in rat pheochromocytoma (PC12; Aβ40) or rat insulinoma (RIN5fm;IAPP) cells. 1a strongly suppressed amyloidogenesis and cell-damagingeffects of Aβ40 (FIGS. 2 a,b,e). Titrations of cytotoxic Aβ40 specieswith 1a yielded an IC₅₀ of 79.8 (+30.3) nM which was nearly identical tothe IC₅₀ of 1 (Table 1). Moreover and in contrast to 1, 1a also stronglysuppressed amyloid self-assembly and cell damage by IAPP; an IC₅₀ of 126(+39.1) nM was determined (FIGS. 2 c,d,e; Table 1). The cyclicconstraint of 1a is thus fully compatible with potent amyloid inhibitoryfunction toward both Aβ40 and IAPP.

TABLE 1 IC₅₀ of inhibitory effects on cell-damaging amyloidself-assembly of Aβ40 or IAPP. IC₅₀ (±SD) IC₅₀ ( ±SD) (nM) (nM) Aβ40IAPP Peptide inhibition^([a]) inhibition^([a]) R3-GI (1) 116 (±11)^([5])n.d.^([b]) 1a 79.8 (±30.3) 126 (±39.1) 2 n.d.^([b]) n.d.^([b]) 2a 125.1(±73.4) 47.6 (±15.5) 2b 654.3 (±227.8) 425.7 (±77.7) 2c 204 (±83.6)n.d.^([b]) 2d 702.6 (±339.3) n.d.^([b]) 2e 542.5 (±240.7) n.d.^([b])^([a]) IC₅₀s, means (±SD) from 3-4 titration assays (n = 3 each) (Aβ40,500 nM; IAPP, 100 nM). ^([b]) n.d., non-determined (non-inhibitor).

To reduce the size of 1, the inventors synthesized its analog 2 lackingregion IAPP(8-13). N-terminal truncation was based on the suggestionthat IAPP(14-18) and IAPP(22-28) mediate key interactions for both IAPPself- and hetero-assembly. However, 2 was unable to block amyloidself-assembly of the two polypeptides (FIGS. 2a-d ).

The inventors hypothesized that conformational restriction of 2 viacyclization might restore inhibitory function and synthesized 2a withtwo flanking disulfide-bridged cysteines (FIG. 1). In fact, 2a blockedAβ40 cytotoxic self-assembly with an IC₅₀ of 125.1 (+73.4) nM which isclose to the IC₅₀ of 1 (FIGS. 2 a,b,e; Table 1). Moreover, 2a blockedIAPP cytotoxic self-assembly as well; an IC₅₀ of 47.6 (±15.5) nM wasdetermined consistent with 2a being even more potent (˜3-fold) than thelonger is (FIGS. 2 c,d,e, Table 1). Interestingly, the far-UV CD spectraof both cyclic and linear peptides had similar shapes and wereindicative of random coil and β-sheet/β-turn contents at ˜1/1 ratio(FIG. 2f ). Differences between inhibitory activities might thus be dueto stabilization of specific folds enabling specific side chaintopologies and high affinity functional interactions with Aβ40 or IAPP.Indeed, fluorescence spectroscopic titrations revealed mid-nanomolarapp. K_(d)s for interactions of cyclic inhibitors is and 2a withN-terminal fluorescently labeled Aβ40 or IAPP as previously found for 1,whereas much weaker binding was found for the non-inhibitor 2.

Recent findings suggest that IAPP residues Phe15, Leu16, Phe23 and Ile26are key residues of IAPP-IAPP and IAPP-Aβ40(42) interactions and thatthe nature of the linker tripeptide determines ISM function. Aiming atminimizing IAPP-derived elements, the inventors next asked if these 4residues and the RRR linker would be sufficient for amyloid inhibitoryfunction and synthesized analog 2b with 7 out of the ii non-Gly residuesof 2a substituted with Gly (except for Cys); only the side chains of theabove 4 residues were maintained (FIG. 1).

Remarkably, 2b strongly suppressed amyloid self-assembly andcell-damaging effects of both Aβ40 (IC₅₀, 654.3 (±227.8) nM) and IAPP(IC₅₀, 425.7 (+77.7) nM) (FIG. 3, Table 1). Such suppressive effect wasnot, or to a much lesser degree, seen for mutant peptides in which oneor several of residues F15, L16, F23 and I26 had been replaced by Ala(data not shown). In addition, fluorescence spectroscopic titrationsyielded mid-nanomolar app. K_(d)s for the interactions of 2b withfluorescently labeled Aβ40 and IAPP which were in a similar range to theIC₅₀s (data not shown). The CD spectrum of 2b exhibited one majorminimum at 230 nm, which was indicative of stabilized turn structures(FIG. 3f ). The 4 IAPP key residues and the RRR tripeptide arrangedwithin the cyclic oligoglycine scaffold of 2b may thus comprise a motifmedia-ting cross-amyloid inhibitor function; moreover, the potentinhibitory effects of 2b toward amyloid self-assembly of both Aβ40 andIAPP make it a lead for anti-amyloid drugs.

Resistance toward plasma proteases is an important requirement for anydrug candidate. Therefore, the inventors determined the proteolyticstabilities of the above peptides in human plasma in vitro using RP-HPLCand MALDI-TOF-MS. Unfortunately, all of them were rapidly degraded;half-life times (to) were <1 h (FIG. 4a ).

To improve the proteolytic stability of 2b, 3 rounds of sequenceoptimization were performed next (FIG. 1). First, analog 2c with 3 D-Argin the linker region instead 3 L-Arg was synthesized followed by 2d inwhich, in addition, Phe and Leu residues were replaced by D-ones.However, to values were only slightly improved (˜1-2 h) (FIG. 4a ).Thus, 2e in which additionally the two L-Cys were replaced by D-Cys wassynthesized. Most importantly, 2e exhibited highly improved proteolyticstability also in comparison to other peptides (to >11 h) being >30-foldmore resistant than 1 (t_(1/2)˜20 min) and >15-fold more resistant thanits L-precursor 2b (t_(1/2)˜45 min) (FIG. 4a ).

The inventors next asked whether switching chiralities may have affected2b structure and function. In fact, the shapes of the CD spectra of2c-2e were similar to 2b but their minima were blue-shifted indicativeof different types of turns and/or β-strand contents (data not shown).However, 2c, 2d, and 2e bound both Aβ40 and IAPP with similar highaffinities to 2b (data not shown). Notably, all three peptides werenanomolar inhibitors of Aβ40 amyloid self-assembly; their IC₅₀ valueswere similar to the IC₅₀ of 2b (FIGS. 3a-e , Table 1). Most remarkably,all of them lost the ability of 2b to block IAPP amyloidogenesis, whichrendered them into Aβ40-selective inhibitors; notably, 2e selectivitywas maintained for up to an at least 50-fold molar excess to IAPP (FIGS.3 c,d,e).

Next, the inventors studied effects of MCIPs on Aβ42 amyloidogenesis. Infact, all of them strongly suppressed formation Aβ42 fibrils andcell-damaging assemblies (FIGS. 4b,c ). Titrations of N-terminalfluorescently labeled Aβ42 (FITC-Aβ42) (5 nM) with MCIPs revealedmid-to-low nanomolar binding affinities (data not shown). Mostimportantly, ex vivo electrophysiological studies in mouse brains showedthat 2a, 2b and 2e ameliorated Aβ42-mediated inhibition of hippocampalsynaptic long term potentiation (LTP) which is linked to loss of memoryand cognitive functions in AD; these results support the physiologicalrelevance of the in vitro results (FIGS. 4d,e ).

Blood-brain-barrier (BBB) crossing is a highly desirable property fordrug candidates targeting the amyloid cascade in AD. However, the highlyrestrictive nature of the BBB allows for only very fewneuropharmaceuticals to be delivered to the brain. MCIP 2e is a quitesmall (<2 kDa) macrocyclic peptide containing an RRR segment, two amidebond N-methylated residues, 4 aromatic/large hydrophobic residues, and 8flexible Gly residues; these are all features linked to membranepermeability. Thus, the inventors next studied whether N-terminalfluorescein-labeled 2e (Fluos-2e) can cross the BBB by using awell-established cell model of human cerebral microvascular endothelialcells (hCMECs) grown as confluent monolayers on Transwell membranes. Infact, Fluos-2e crossed the monolayer with an apparent permeability(P_(app)) at 2 h of 14.6×10⁻⁶ (±3.36) cm/s (mean (±SEM), n=3), which issimilar to the P_(app) of other BBB crossing peptides (FIGS. 4f,g ). Ofnote, hCMECs express substantial surface levels of the prominent brainprotease neprilysin and 2e was fully resistant to degradation in an invitro human neprilysin digestion assay (data not shown).

The present inventors were thus able to design and produce highly potentamyloid inhibitors of both Aβ40(42) and IAPP, or of Aβ40(42) alone. Theyalso showed that the chirality of these inhibitors controls inhibitorselectivity. Furthermore, a systematic sequence optimization led toinhibitor 2e, which is a nanomolar Aβ40(42)-selective inhibitor whichexhibits high proteolytic stability in human plasma and humanblood-brain-barrier crossing ability in a cell model which are twohighly desirable properties for anti-amyloid drugs in Alzheimer'sdisease.

The invention claimed is:
 1. A peptide having an amino acid sequenceaccording to formula 0

wherein Z1 and Z2 are selected from the following pairs a) cysteine andcysteine, b) aspartic acid and lysine, or lysine and aspartic acid, c)aspartic acid and ornithine, or ornithine and aspartic acid, d) asparticacid and 2,4-diaminobutyric acid, or 2,4-diaminobutyric acid andaspartic acid, e) aspartic acid and 2,3-diaminopropionic acid, or2,3-diaminopropionic acid and aspartic acid, f) glutamic acid andlysine, or lysine and glutamic acid, g) glutamic acid and ornithine, orornithine and glutamic acid, h) glutamic acid and 2,4-diaminobutyricacid, or 2,4-diaminobutyric acid and glutamic acid, i) glutamic acid and2,3-diaminopropionic acid, or 2,3-diaminopropionic acid and glutamicacid; with

denoting a covalent bond between Z1 and Z2, thus providing for acyclization of the peptide; X1, X2, X3, X4, X5, X6, and X7 are,independently at each occurrence, selected from glycine, asparagine,valine, histidine, leucine, serine, alanine, and threonine; F is,independently at each occurrence, phenylalanine; L is leucine; U is,independently at each occurrence, selected from arginine, homoarginine,citrulline, ornithine, lysine, and norleucine; G is glycine; I isisoleucine; wherein Z1, Z2, X1-X7, F, L, U, G and I are L-amino acidresidues or D-amino acid residues, or some of Z1, Z2, X1-X7, F, L, U, Gand I are L-amino acid residues and others are D-amino acid residues;and pharmaceutically acceptable salts, esters, solvates, polymorphs andmodified forms thereof.
 2. The peptide according to claim 1, having anamino acid sequence according to formula 1

or an amino acid sequence according to formula 1*

wherein C is cysteine; X1, X2, X3, X4, X5, X6, and X7 are, independentlyat each occurrence, selected from glycine, asparagine, valine,histidine, leucine, serine, alanine, and threonine; F is, independentlyat each occurrence, phenylalanine; L is leucine; R is arginine; G isglycine; I is isoleucine;

is a disulfide bond;

is N-methyl; wherein C, X1-X7, F, L, R, G and I are L-amino acidresidues or D-amino acid residues, or some of C, X1-X7, F, L, R, G and Iare L-amino acid residues and others are D-amino acid residues; andpharmaceutically acceptable salts, esters, solvates, polymorphs andmodified forms thereof.
 3. The peptide according to claim 1, whereineither a) X1 and X4 are asparagine, X2 is valine, X3 is histidine, X4 isglycine, X5 is glycine, X6 and X7 are glycine; b) X1-X7 are glycine,alanine or serine; c) X1-X7 are glycine; d) X1-X3 are glycine, X4 isasparagine, X5 is alanine, X6-X7 are glycine; or e) X1-X3 are glycine,X4 is asparagine, X5 is alanine, X6 is leucine, X7 is serine.
 4. Thepeptide according to claim 1, wherein Z1, Z2, C, X1-X7, F, L, U, R, G,and I are L-amino acid residues.
 5. The peptide according to claim 1,wherein R is, at each occurrence, D-arginine, and/or wherein F is, ateach occurrence, D-phenylalanine, and/or wherein L is D-leucine, and/orwherein Z1, Z2 and C are D-amino acid residues, and/or wherein I isD-isoleucine or N-methyl-D-isoleucine.
 6. The peptide according to claim1, having a sequence according to a formula selected from the followingformulae 2a-2e, and 2a*-2e*:

wherein upper case letters represent L-amino acid residues or D-aminoacid residues, and wherein lower case letters represent D-amino acidresidues.
 7. The peptide according to claim 6, having a sequenceaccording to a formula selected from 2e and 2e*


8. The peptide according to claim 1, wherein said peptide consists of asequence according to any of formulae 0, 0a, 1, 1*, 2a-2e, and 2a*-2e*:


9. The peptide according to claim 1, wherein said peptide is an amyloidinhibitory peptide that binds to Aβ40(42) and/or to islet amyloidpolypeptide (IAPP).
 10. A pharmaceutical composition comprising apeptide according to claim 1 and a pharmaceutically acceptableexcipient.
 11. A kit for the in-vitro or in-vivo detection of amyloidfibrils or aggregates, or for the diagnosis of Alzheimer's disease ortype 2 diabetes in a patient, said kit comprising the peptide accordingto claim 1, in a freeze-dried form in a suitable container, a bufferedsolvent in a separate container for reconstitution of said peptide insolution, and, optionally, means to dispense said peptide oncereconstituted in solution.
 12. The peptide, according to claim 1,wherein said peptide has an amino acid sequence according to formula 0a

wherein Z1, Z2, X1-X7, F, L, U, G, I are as defined in claim 1, and

is N-methyl.
 13. The peptide according to claim 6, wherein upper caseletters represent L-amino acid residues and lower case letters representD-amino acid residues.
 14. The peptide according to claim 7, whereinupper case letters represent L-amino acid residues and lower caseletters represent D-amino acid residues.